Publications (37) View all
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Article: Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage.
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ABSTRACT: BACKGROUND: Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells. METHODS: A549 cells were irradiated with γ-rays (2.0Gy) or UVB (5-10mJ/cm2). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin-luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection. RESULTS: γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells. CONCLUSION: Our results suggest that activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation. General significance Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.Biochimica et Biophysica Acta 02/2013; · 4.66 Impact Factor -
Article: Involvement of P2Y11 receptor in IFN-γ-induced IL-6 production in human keratinocytes.
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ABSTRACT: Extracellular ATP and P2 receptors are reported to be involved in interleukin-6 (IL-6) production by human keratinocytes, but the role of extracellular ATP in cytokine-induced IL-6 production remains unclear. In this study, we investigated the involvement of various P2 receptors in IL-6 production induced by the Th1 cytokine interferon-gamma (IFN-γ in a human keratinocyte cell line, HaCaT. IFN-γ increased IL-6 production in HaCaT cells. A non-selective antagonist of P2Y receptors (suramin), a selective P2Y11 receptor antagonist (NF157), ecto-nucleotidase (apyrase), and a soluble adenylate cyclase inhibitor (KH7) all inhibited IL-6 production. It was further confirmed that ATP was released from HaCaT cells stimulated with IFN-γ. These results suggest that extracellular ATP and P2Y11 receptor are involved in IFN-γ-induced IL-6 production. Knockdown of P2Y11 receptor suppressed IL-6 production, strongly supporting this idea. In conclusion, these data demonstrate that P2Y11 receptor mediates IFN-γ-induced IL-6 production in human keratinocytes, and suggest the importance of purinergic signaling in IFN-γ-induced skin inflammatory conditions, such as psoriasis.European journal of pharmacology 02/2013; · 2.59 Impact Factor -
Article: P2X4 receptor regulates P2X7 receptor-dependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells.
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ABSTRACT: Activation of P2X7 receptor of dendritic cells plays a significant role in inflammation through production of cytokines such as IL-1β, and recent studies have suggested structural and functional interactions of P2X7 receptor with P2X4 receptor in macrophages. However, it is unknown whether P2X4 receptor modulates P2X7 functions in dendritic cells. Here, we present evidence that expression of P2X4 receptor is required for P2X7 receptor-dependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells (BMDCs). We confirmed expression of both P2X7 receptor and P2X4 receptor in BMDCs. Treatment of BMDCs with 3 mM ATP caused a transient, P2X4-dependent elevation, or spike, of intracellular Ca(2+) level ([Ca(2+)](i)), followed by the sustained P2X7-dependent increase of [Ca(2+)](i). We performed knockdown of P2X4 receptor in BMDCs by transfection with short hairpin RNA targeting this receptor. The ATP-induced initial peak of [Ca(2+)](i) was decreased in P2X4-knockdown cells (P2X4-KD). Further, we found that ATP-induced IL-1β and IL-18 release from LPS-primed BMDCs was suppressed by pretreatment with P2X7 antagonist A438079 or P2X4 antagonist TNP-ATP. The P2X7-dependent IL-1β and IL-18 release was significantly lower in P2X4-KD cells. Chelation of intracellular Ca(2+) also caused suppression of ATP-induced IL-1β and IL-18 release. These results suggest that P2X4 receptor-induced Ca(2+) influx is required for effective production of IL-1β and IL-18 via activation of P2X7 receptor in BMDCs. We conclude that co-expression of P2X4 receptor with P2X7 receptor in dendritic cells leads to enhancement of inflammation through facilitation of P2X7-dependent release of pro-inflammatory cytokines.Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor -
Article: Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor.
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ABSTRACT: It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3-24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.PLoS ONE 01/2013; 8(4):e59778. · 4.09 Impact Factor -
Article: Feasibility study of B16 melanoma therapy using oxidized ATP to target purinergic receptor P2X7.
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ABSTRACT: The P2X7 receptor is not only involved in cell proliferation, but also acts as an adenosine 5'-triphosphate (ATP)-gated non-selective channel, and its expression is increased in human melanoma. An irreversible antagonist of P2X7, such as oxidized ATP (oxATP), might block P2X7 receptor-mediated ATP release and proliferative signaling. Therefore, we carried out basic studies to test this idea and to examine the feasibility of using oxATP to treat B16 melanoma. We first found that low-pH conditions (mimicking the hypoxia and acidosis commonly seen in solid tumors) induced P2X7 receptor-mediated ATP release from B16 melanoma cells. Then, we compared the proliferation rates of B16 melanoma wild-type cells and B16 P2X7 receptor-knockdown clone (P2X7-KDC) cells in the presence of P2X7 agonists. The proliferation rate, as well as the ATP release, of agonist-treated P2X7-KDC cells was lower than that of agonist-treated wild-type cells. Next, the effect of P2X7 antagonist oxATP on B16 melanoma cell growth was examined in vitro and in vivo. oxATP significantly decreased B16 melanoma cell proliferation in vitro, and also significantly inhibited tumor growth in B16 melanoma-bearing mice. These data indicate that extracellularly released ATP may serve as an intercellular signaling molecule. We propose that the P2X7 receptor is a promising target for treatment of solid tumors.European journal of pharmacology 09/2012; 695(1-3):20-6. · 2.59 Impact Factor
